Tina Jaeger

YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

MurQ Etherase Is Required by Escherichia coli in Order To Metabolize Anhydro-N-Acetylmuramic Acid Obtained either from the Environment or from Its Own Cell Wall

MurQ is an N-acetylmuramic acid-phosphate (MurNAc-P) etherase that converts MurNAc-P to N-acetylglucosamine-phosphate and is essential for growth on MurNAc as the sole source of carbon (T. Jaegar, M. Arsic, and C. Mayer, J. Biol. Chem. 280:30100-30106, 2005). Here we show that MurQ is the only MurNAc-P etherase in Escherichia coli and that MurQ and AnmK kinase are required for utilization of anhydro-MurNAc derived either from cell wall murein or imported from the medium.

Scission of the Lactyl Ether Bond of N-Acetylmuramic Acid by Escherichia coli “Etherase”

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique d-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385–2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and d-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3–O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (β-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

Identification of a Phosphotransferase System of Escherichia coli Required for Growth on N-Acetylmuramic Acid

We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIA(Glc)), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His(6) fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

N-acetylmuramic acid 6-phosphate lyases (MurNAc etherases): role in cell wall metabolism, distribution, structure, and mechanism.

Abstract.MurNAc etherases cleave the uniqued-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate- binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: β-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added.

LadS is a calcium-responsive kinase that induces acute-to-chronic virulence switch in Pseudomonas aeruginosa.

Virulence of pathogenic bacteria is a tightly controlled process to facilitate invasion and survival in host tissues. Although pathways controlling virulence have been defined in detail, signals modulating these processes are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa causes acute and chronic infections in humans. Disease progression is typically associated with a loss of acute virulence and the emergence of biofilms and chronic behaviour. The acute-to-chronic switch is governed by the global Gac/Rsm pathway. Using a newly developed acute–chronic dual reporter system we show that calcium stimulates the Gac/Rsm pathway via the Gac-associated hybrid histidine kinase LadS. We show that calcium binds to the periplasmic DISMED2 sensor domain of LadS to activate its kinase activity. Activation of the Gac/Rsm pathway by calcium leads to a switch to the chronic program and confers drug tolerance by reducing P. aeruginosa growth rate. Clinical isolates from cystic fibrosis airways retain their calcium response during chronic infections. Our data imply that calcium sensing evolved as an adaptation to the opportunistic lifestyle of P. aeruginosa and that calcium serves as a host signal to balance acute-to-chronic behaviour during infections. Establishing calcium signalling in host–pathogen interaction adds to growing evidence indicating key roles for calcium in bacterial signalling.

Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms

Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.